N. Okishio et al., FISSION YEAST NDA1 AND NDA4, MCM HOMOLOGS REQUIRED FOR DNA-REPLICATION, ARE CONSTITUTIVE NUCLEAR PROTEINS, Journal of Cell Science, 109, 1996, pp. 319-326
The ndal(+) and nda4(+) genes of the fission yeast Schizosaccharomyces
pombe encode proteins similar to budding yeast MCM2 and MCM5/CDC46, r
espectively, which are required for the early stages of DNA replicatio
n. The budding yeast Mcm proteins display cell-cycle dependent localiz
ation. They are present in the nucleus specifically from late M phase
until the beginning of S phase, so that they were suggested to be comp
onents of a replication licensing factor, a positive factor for the on
set of replication, which is thought to be inactivated after use, thus
restricting replication to only once in a cell cycle. In the present
study, we raised antibodies against Nda1 or Nda4 and identified 115 kD
a and 80 kDa proteins, respectively. Their immunolocalization was exam
ined in wild-type cells and in various cell-cycle mutants. Both Nda1 a
nd Nda4 proteins remained primarily in the nucleus throughout the cell
cycle. In mutants arrested in G(1), S, and G(2) phases, these protein
s were also enriched in the nucleus. These results indicate that the d
ramatic change in subcellular localization as seen in budding yeast is
not essential in fission yeast for the functions of Nda1 and Nda4 pro
teins to be executed. The histidine-tagged nda1(+) gene was constructe
d and integrated into the chromosome to replace the wild-type nda1(+)
gene. The resulting His-tagged Nda1 protein was adsorbed to the Ni-aff
inity column, and co-eluted with the untagged Nda4 protein, suggesting
that they formed a complex.